This invention relates to a synthetic peptidic substrate having a color-producible and fluorescence-emittable group suitable for the determination of trypsin and .alpha..sub.1 -antitrypsin. The substrate of this invention is remarkably high in the selectivity compared with known substrates and thus suitable for the determination of trypsin. Further, the substrate of this invention can be used for studies of reactions wherein the function of trypsin is inhibited or trypsin is consumed, or studies of factors pertaining to these reactions, particularly for the determination of .alpha..sub.1 -antitrypsin.
.alpha..sub.1 -antitrypsin (hereinafter referred to as ".alpha..sub.1 -AT") is a glycoprotein having a molecular weight of about 51,000 and contained in normal serum in a concentration of 160 to 350 mg/dl. A major function of .alpha..sub.1 -AT is to combine with proteases such as trypsin, chymotrypsin, thrombin, kallikrein, etc., and further with elastase derived from white blood cells, collagenase, etc. to form stable complexes, which results in neutralizing or inhibiting the functions of these proteases.
.alpha..sub.1 -AT is also called as an acute phase reactant and increases remarkably its content in serum in acute inflammation diseases, after surgical operations or destruction of tissues. The content of .alpha..sub.1 -AT is also increased by administration of estrogen or insulin, and in pregnancy, but decreased by serious liver function disorder such as liver cirrhosis, malignant hepatitis and low nutritive conditions. Further, since it has recently been known that the .alpha..sub.1 -antitrypsin content is also increased by a malignant tumor, .alpha..sub.1 -AT is expected to be used as a marker for related tumors.
Specificity of qualitative change of .alpha..sub.1 -AT has been noticed since a report of complication of pulmonary emphysema and an .alpha..sub.1 -antitrypsin deficiency [S. Eriksson: Acta Medica Scandinavica, Supplementum 432, pp. 41-75 (1965)]. Since that time, a relationship between .alpha..sub.1 -AT and chronic obstructive lung disease has gradually become clear. Therefore, it is significant that .alpha..sub.1 -AT which has been noticed clinically can be determined simply and precisely.
The determination of .alpha..sub.1 -AT in serum can be carried out by measuring inhibitory ability of trypsin activity. That is, when trypsin is added to serum, a part of the trypsin added is deactivated depending on the amount of .alpha..sub.1 -AT. Thus, the activity of remaining trypsin is measured to determine .alpha..sub.1 -antitrypsin.
As substrates for determining trypsin, there were used formerly proteins such as gelatin, hemoglobin, etc. But after Bergman et al reported that trypsin had amidase and esterase functions in addition to the protein decomposition function in 1939, various synthetic substrates (e.g. Bz-.sub.L -Arg-OEt, Tos-.sub.L -Arg-OMe, Bz-.sub.DL -Arg-pNA, Tos-.sub.L -Arg-pNA, etc.) have been used in the determination based on the amido bond or the ester bond of arginine. But these synthetic substrates had problems in the specificity of substrates and solubility in water or buffer solutions. As substrates for determining trypsin, there have further been developed Z-Val-Gly-Arg-pNA (CHR-TRY, a trade name, mfd. by Pentapharm A.G.), etc. (U.S. Pat. No. 4,278,762) and aminomethylcoumarin (AMS) which can emit fluorescence after freed [Smith et al.: Chemical Abstr. 92: 159413h (1980)]. In the above, Bz means benzoyl, Arg araginyl, OEt ethoxy, Tos p-toluenesulfonyl, OMe methoxy, pNA para-nitroanilide, Z a benzyloxycarbonyl group, Val valyl, and Gly glycyl.
On the other hand, in the determination of .alpha..sub.1 -AT in serum by using a color-developable substrate, if cross reactions take place with proteases other than trypsin such as plasmin, thrombin, factor Xa, urokinase, etc. which are expected to be present in the serum, precise measurement cannot be expected.
The above-mentioned CHR-TRY which is the best substrate for the determination of trypsin is not always satisfactory in the substrate specificity. That is, it is known that CHR-TRY reacts considerably with thrombin, plasmin, factor Xa, urokinase, etc. other than trypsin. Further, in the process of colorimetric determination of a yellow color of p-nitroanilide produced by using CHR-TRY as a substrate, influences of serum components cannot be prevented.
The amount of .alpha..sub.1 -AT in serum is usually as large as 160 to 350 mg/dl, which value is about 10 times as large as that of .alpha..sub.2 -macroglobulin (.alpha..sub.2 -M) in a molar concentration, .alpha..sub.2 -M being also present in serum. Further, .alpha..sub.1 -AT easily reacts with various inflammation stimuli to increase the amount thereof about 2 to 4 fold in the serum. In order to determine .alpha..sub.1 -AT which is present in a large amount and changes in amounts greatly in the following method, there are many problems: ##STR2##
That is, in order to measure .alpha..sub.1 -AT precisely with a serum dilution rate of about 100 to 150 times usually used in this art, a large amount of trypsin should be used together with a considerably large amount of substrate. In contrast, in order to carry out the assay by using suitable amounts of the enzyme, and substrate in a suitable measuring range, it is necessary to dilute the serum about 500 to 1000 times. But such a high dilution rate is undesirable since procedures in daily examination becomes complicated and easily causes errors in measurement, the reactivity with .alpha..sub.1 -AT and trypsin changes, influence of .alpha..sub.2 -macroglobulin (.alpha..sub.2 -M) becomes not negligible, and precise measurement becomes difficult. In order to make the serum dilution rate about 100 to 150 times, it is necessary to develop new substrates which have proper reactivity with trypsin and high solubility in water or buffer solutions.